This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. A major goal of the Rossie lab is to determine the substrates for the serine/threonine phosphatase protein phosphatase 5 (PP5). Heat shock protein 90 (Hsp90), the major cellular binding partner for PP5, is a molecular chaperone responsible for the folding and maturation of numerous cellular proteins. The role of PP5 in Hsp90 chaperone complexes is not understood. In this project, we will define the role and targets of PP5 in the Hsp90 chaperone complex. We will isolate PP5 complexes from cells expressing wild-type PP5, catalytically inactive PP5 or a mutant form of PP5 unable to bind Hsp90. Using mass spectrometry centric capabilities in the Center, we will identify protein partners that are co-immunoprecipitated with PP5. Partners of the PP5 mutant that cannot bind Hsp90 are expected to represent PP5 partners unrelated to the Hsp90 complex. We will determine the phosphorylation state of Hsp90 and other proteins in complexes containing either wild-type or inactive PP5 to determine which members of the Hsp90 complex undergo changes in phosphorylation as a function of PP5 activity and identify sites of PP5 dephosphorylation. Since PP5 is complexed with glucocorticoid receptors (GRs) together with Hsp90, GRs can be activated by phosphorylation events and are negatively regulated by PP5. We will also specifically evaluate the phosphorylation status of GRs. Finally we will analyze the composition of PP5:Hsp90 complexes from rat brain in order to verify that the protein partners seen in our cell studies also exist in native tissue containing normal levels of PP5. Specific Aims include: 1. To identify PP5 binding partners present in the PP5-Hsp90 complexes. 2. To determine whether PP5 dephosphorylates mammalian Hsp90, co-chaperones and/or clients, and identify sites of phosphorylation that change as a function of PP5 activity. 3. To determine if GRs are dephosphorylated by PP5 in PP5-Hsp90 complexes. 4. To identify proteins present in native PP5-Hsp90 complexes isolated from rat brain.